Herein we report a combined electrochemical and ESI-MS study of the enzymatic hydrolysis efficiency of DNA self-assembled monolayers (SAMs) on gold, platform systems for understanding nucleic acid surface chemistry and for constructing DNA-based biosensors. Our electrochemical approach is based on the comparison of the amounts of surface-tethered DNA nucleotides before and after Exonuclease I (Exo I) incubation using electrostatically bound [Ru(NH3)6]3+ as redox indicators. It is surprising to reveal that the hydrolysis efficiency of ssDNA SAMs does not depend on the packing density and base sequence, and that the cleavage ends with surface-bound shorter strands (9-13 mers). The ex-situ ESI-MS observations confirmed that the hydrolysis products for ssDNA SAMs (from 24 to 56 mers) are dominated with 10-15 mer fragments, in contrast to the complete digestion in solution. Such surface-restrained hydrolysis behavior is due to the steric hindrance of the underneath electrode to the Exo I/DNA binding, which is essential for the occurrence of Exo I-catalyzed processive cleavage. More importantly, we have shown that the hydrolysis efficiency of ssDNA SAMs can be remarkably improved by adopting long alkyl linkers (locating DNA strands further away from the substrates).